The influence of two process parameters (starter and fermentation period) on sourdough bread qualities was investigated. Bifidobacterium longum/Enterococcus faecium/Lactobacillus acidophilus (a mixed culture) was used as a starter. The five production conditions tested were: Control (sourdough fermentation with yeast); LAB 1(fermentation with mixed culture); LAB 2 (fermentation with yeast and mixed culture, respectively); LAB 3 (fermentation with yeast and mixed culture); and LAB 4 (first fermentation with yeast and mixed culture, respectively, followed by a second fermentation using the combination). The LAB 4 process showed the most favorable fermentation characteristics upon CrumbScan analysis, and the highest specific bread volume (5.14 mL/g). These results were reflected in the sensory evaluation of bread produced by the LAB 4 process; the bread achieved an excellent overall acceptance ranking of 3.7. Upon firmness analysis, the LAB 2, LAB 3, and LAB 4 bread figures were 113.67 g, 111.97 g, and 113.50 g, respectively. Thus, the firmness of LAB 2, LAB 3, and LAB 4 bread was higher than that of the control (93.20 g), although the aroma compounds of bread produced by the five processes did not differ. These results show that LAB 4 bread had improved sourdough properties, compared to control.